Oxidative damage to biomolecules by the hydroxyl radical generated by metals in Fenton and Fenton-like reactions has been widely demonstrated in vitro, as measured by lipid peroxidation and DNA damage, but has not been proven to occur in vivo. The proposed generation of hydroxyl radical by such chemistry in vivo was investigated using the ESR technique of spin trapping. A procedure was developed from which the spin-trapped adducts of free radical metabolites, generated in vivo under conditions of iron overload, are detected in the bile of rats. These studies demonstrate that the generation of hydroxyl radical from simple FE(II) Fenton chemistry can indeed occur in vivo. Cu(I) also exhibits strong Fenton-like chemistry in vitro. Our results show, however, that this chemistry is not operative for copper in the presence of a wide variety of thiols, including cysteine and glutathione. These results explain the lack of detection in bile of any significant amount of free radical metabolites formed in vivo, even during conditions of severe copper overload. This is a significant observation as it may partially explain why and how the body bypasses the need for sequestering free copper in vivo as it does for iron (i.e., in ferritin). Co(II), unlike FE(II) and Cu(I), does not easily generate free hydroxyl radical in vitro. Consequently, preliminary experiments have failed to detect any spin-trapped free radical metabolites in vivo. Certain Co(II) chelates, however, do form highly oxidative complexes with oxygen and may cause oxidative damage in vivo without involving free hydroxyl radical. Other complexes of Co(II) generate significant amounts of superoxide from hydrogen peroxide in vitro.